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目的:建立使用高效液相色谱-电化学法测定大鼠下丘脑、海马、皮质中单胺类神经递质及其代谢产物的方法,所测指标包括去甲肾上腺素(NE)、五羟吲哚乙酸(5-HIAA)、高香草酸(HVA)、多巴胺(DA)、和5-羟色胺(5-HT)。方法:色谱柱采用Waters C18反相色谱柱(3.9mm×150mm,5μm),流动相为水相(每1L中含辛烷磺酸钠2.5g,磷酸二氢钾13.6g,乙二胺四乙酸36mg,pH值3.3):甲醇:乙腈=78:19:3,流速为0.8mL/min,柱温35℃;电化学检测器工作电极为玻璃碳电极,参比电极为银/氯化银(Ag/AgCl)电极,工作电压为0.7V[1]。结果:脑组织中五种神经递质的保留时间分别为:去甲肾上腺素3.4min、五羟吲哚乙酸4.2min,高香草酸4.8min,多巴胺5.8min,5-羟色胺11.4min;最低检测限为4～16ng/mL。线性范围为20～320ng。脑组织样品的相对回收率为87.5%～113.9%,日内、日间精密度RSD小于7%。结论:该方法具有快速、准确、灵敏且样品制备简便等优点,可用于大鼠部分脑区内单胺类神经递质的分离测定。
OBJECTIVE: To establish a method for the determination of monoamine neurotransmitters and their metabolites in rat hypothalamus, hippocampus and cortex using high performance liquid chromatography-electrochemistry. The measured indexes include norepinephrine (NE), pentoxine (5-HIAA), homovanillic acid (HVA), dopamine (DA), and serotonin (5-HT). Methods: A Waters C18 reversed-phase column (3.9 mm × 150 mm, 5 μm) was used with a mobile phase of water (2.5 g sodium octane sulfonate per liter, 13.6 g potassium dihydrogen phosphate, 36mg, pH 3.3): methanol: acetonitrile = 78: 19: 3, the flow rate of 0.8mL / min, the column temperature of 35 ℃; electrochemical detector working electrode is a glassy carbon electrode, the reference electrode is silver / silver chloride Ag / AgCl) electrode with a working voltage of 0.7V [1]. Results: The retention time of five neurotransmitters in brain tissue were: norepinephrine 3.4min, pentoxynoacetic acid 4.2min, homovanillic acid 4.8min, dopamine 5.8min, serotonin 11.4min; the lowest detection limit 4 ~ 16ng / mL. The linear range is 20 ~ 320ng. The relative recovery of brain tissue samples was 87.5% -113.9%, and the intra-day and inter-day RSDs were less than 7%. Conclusion: The method is rapid, accurate, sensitive and simple preparation of samples. It can be used for the determination of monoamine neurotransmitters in some brain regions of rats.